U.S. patent number 10,238,676 [Application Number 15/036,494] was granted by the patent office on 2019-03-26 for application of ginsenoside rg3 in preparing medicine for preventing and/or treating dementia, and medicine for treating dementia.
This patent grant is currently assigned to Li Fu. The grantee listed for this patent is Li Fu. Invention is credited to Li Fu, Qiang Fu, Xin Gai, Zhengxian Liu, Qi Lu, Kaiqian Wang.
United States Patent |
10,238,676 |
Fu , et al. |
March 26, 2019 |
Application of ginsenoside RG3 in preparing medicine for preventing
and/or treating dementia, and medicine for treating dementia
Abstract
The present invention discloses an application of ginsenoside
Rg3 in preparing a medicine or product for preventing and treating
dementia, and a preparation method thereof, which belong to the
fields of medicines and health care products, and its products
comprise a topical preparation, oral preparation and injection of
the ginsenoside Rg3; and the Rg3 medicine or formulating prepared
Rg3 compound medicine is prepared by using a solubilizer, a
transdermal agent, a reagent promoting the absorption by
penetrating a blood-brain barrier and an extractive, achieving the
effect that the medicine penetrates the blood-brain barrier, and is
used for preventing and treating dementia.
Inventors: |
Fu; Li (Dalian, CN),
Wang; Kaiqian (Dalian, CN), Fu; Qiang (Dalian,
CN), Liu; Zhengxian (Dalian, CN), Lu;
Qi (Dalian, CN), Gai; Xin (Dalian,
CN) |
Applicant: |
Name |
City |
State |
Country |
Type |
Fu; Li |
Dalian |
N/A |
CN |
|
|
Assignee: |
Fu; Li (Dalian,
CN)
|
Family
ID: |
53178919 |
Appl.
No.: |
15/036,494 |
Filed: |
November 6, 2014 |
PCT
Filed: |
November 06, 2014 |
PCT No.: |
PCT/CN2014/090475 |
371(c)(1),(2),(4) Date: |
May 13, 2016 |
PCT
Pub. No.: |
WO2015/074494 |
PCT
Pub. Date: |
May 28, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160271154 A1 |
Sep 22, 2016 |
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Foreign Application Priority Data
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Nov 22, 2013 [CN] |
|
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2013 1 0589909 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
25/28 (20180101); A61K 36/236 (20130101); A61K
36/42 (20130101); A61K 36/9066 (20130101); A61K
36/888 (20130101); A61K 31/704 (20130101); A61K
36/258 (20130101); A61K 36/258 (20130101); A61K
2300/00 (20130101); A61K 36/236 (20130101); A61K
2300/00 (20130101); A61K 36/888 (20130101); A61K
2300/00 (20130101); A61K 36/42 (20130101); A61K
2300/00 (20130101); A61K 36/9066 (20130101); A61K
2300/00 (20130101) |
Current International
Class: |
A61K
31/704 (20060101); A61K 36/9066 (20060101); A61K
36/888 (20060101); A61K 36/258 (20060101); A61K
36/236 (20060101); A61K 36/42 (20060101) |
Field of
Search: |
;514/26 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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101031580 |
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Sep 2007 |
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CN |
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101133075 |
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Feb 2008 |
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CN |
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101312650 |
|
Nov 2008 |
|
CN |
|
Other References
The Merck Manual, 16th Edn. 1992, pp. 1403-1407. cited by examiner
.
Yang et al, J. Pharmacy and Pharmacology, 2009, 61, 375-380. cited
by examiner .
Liu et al, Journal of Traditional Chinese Medicine, Aug. 15, 2013,
33(4), 449-454. cited by examiner .
International Search Report for International application No.
PCT/CN2014/090415, dated Feb. 2, 2015. cited by applicant .
Wang, R., "Neuroprotective Effects and Brain Transport of
Ginsenoside Rg1", ScienceDirect, Chinese Journal of Natural
Medicines, 2009, pp. 315-320, vol. 7, No. 4. cited by applicant
.
Hou, J. "Ginsenoside Rg3 Prevents Oxidative Stress-Induced
Astrocytic Senescence and Ameliorates Senescence Paracrine Effects
on Glioblastoma", Molecules, 2017, pp. 1-14, 22, 1516. cited by
applicant.
|
Primary Examiner: Krishnan; Ganapathy
Attorney, Agent or Firm: Greer, Burns & Crain, Ltd.
Claims
The invention claimed is:
1. A ginsenoside Rg3 composition consisting of 20(R)-ginsenoside
Rg3 with a purity of 98%, Rhizoma Chuanxiong volatile oil, 60%
ethanol, polyethylene glycol 200, glycerol, and water, and the
weight or volume ratio of the 20(R)-ginsenoside Rg3 with the purity
of 98%, Rhizoma Chuanxiong volatile oil, 60% ethanol, polyethylene
glycol 200, and glycerol is 1 g:1 g:10 ml:5 ml:20 g.
2. A method of producing the ginsenoside Rg3 composition of claim
1, comprising the following steps: weighing accurately 1 g of
ginsenoside Rg3 with a purity of 98% and 1 g of Rhizoma Chuanxiong
oil, adding 10 ml of 60% ethanol, 5 ml of polyethylene glycol 200,
and 20 g of glycerol, heating the mixture, stirring to dissolve the
mixture, and adding water to obtain the ginsenoside Rg3
composition.
3. A method of treating Alzheimer's disease, comprising a step of
administering an effective amount of the ginsenoside Rg3
composition of claim 1 to a patient in need.
Description
This application is a U.S. National Phase under 35 U.S.C. .sctn.
371 of International Application No. PCT/CN2014/090475, filed Nov.
6, 2014, which claims priority under 35 U.S.C. .sctn..sctn. 119 and
365 to Chinese Application No. 201310589909.0, filed Nov. 22,
2013.
TECHNICAL FIELD
The present invention relates to a ginsenoside Rg3 pharmaceutic
preparation and a preparation method and application thereof, which
belong to the field of medicines.
BACKGROUND ART
When entering the 21.sup.st century, with the aging of the
population, the morbidity of senile dementia is increasing year by
year in the world. There are about 20 million people suffering from
senile dementia around the world as reported, wherein there are
about 5-7 million people in China. The number of people who died
because of suffering from senile dementia is in the fourth place
after heart disease, cancer and stroke. In China, the morbidity of
dementia of people over 65 years old is 6-7%; the morbidity of
dementia of people over 70 years old is 13-15%; the morbidity of
dementia of people over 80 years old is 22-25%; unfortunately,
there are no better methods to effectively treat and prevent the
senile dementia.
The senile dementia comprises Alzheimer's disease (AD) and vascular
dementia (VD), the modern medical science has not figured out the
pathogenesis of AD, and it is currently considered to be a
progressive and neuro-degenerative disease. However, the VD is
various dementias resulted from neurocranium internal hemorrhage,
multiple infarct, arteriosclerosis encephalopathy acute brain
trauma, encephalic space-occupying lesion, alcoholic intoxication,
brain nutritional deficiency and metabolic endocrine disorder.
No matter what results in the senile dementia, the early symptoms
are all forgetfulness, and hypesthesia of computational power and
language competence; the second phase is serious memory loss,
direction loss and of few words; and the third phase is serious
thinking capacity disorder and vague orientation. Therefore, for
studying the prevention and treatment of the senile dementia, it is
prime to prevent memory and intelligence decline, and the
ginsenoside Rg3 has significant effects of improving memory and
delaying the ageing, which is also one of objectives of the present
invention.
At present, a large number of medical research materials prove that
the cerebral cortex and hippocampal cholinergic neurons of people
suffering from senile dementia are largely lost, the presynaptic
specific cholinergic mark choline acetyl transferase (ChAT) of a
cholinergic transfer medium acetyl choline (Ach) is remarkably
decreased, and this is one of disease causes of the senile
dementia, so how to increase the functions of a reticular formation
ascending activating system of the brain stem and promote Ach
synthesis is an effective method for preventing and treating the
senile dementia. Ginsenoside Rg3 can effectively activate the
functions of the reticular formation ascending activating system of
the brain stem, promote the Ach synthesis, increase cerebral blood
flow, improve microcirculation, and therefore can be used for
preventing and treating the senile dementia, and this is also the
second objective of the present invention.
At present, the medicines used for preventing and treating the
dementia mainly include donepezil, rivastigmine, galantamine,
piracetam, aniracetam, vinpocetine, nicergoline, dihydroergotoxine,
huperaine A, ginkgo leaves agent, brain protein hydrolysate,
citicoline, cholinesterase inhibitor, xanthine, nonsteroidal
anti-inflammatory medicine, estrogen, etc. Although these medicines
have a certain relieving effect, but side effects are great,
limiting their general use.
Chinese medicines for treatment of senile dementia are as follows:
firstly, on the aspect of treatment from the standpoint of liver
and kidney, body essence and marrow supplementing prescriptions are
mostly adopted, such as Radix Rehmanniae Preparata, Fructus Corni,
Rhizoma Polygonati, Carapax et Plastrum Testudinis, Colla Corii
Asini, Fructus Lycii, Cornu Cervi Pantotrichu, etc; secondly, on
the aspect of treatment through phlegm, the prescriptions for
invigorating spleen to eliminate dampness, eliminating phlegm,
inducing resuscitation and restoring consciousness are mostly
adopted, such as Radix Curcumae, Rhizoma Acori Tatarinowii, Radix
Polygalae, Fructus Alpiniae Oxyphyllae, XINGSHEN JIAONANG and the
like;
thirdly, on the aspect of treatment through QI-stagnation and blood
stasis, the prescriptions for regulating QI, activating blood
circulation, inducing resuscitation and restoring consciousness are
mostly adopted, such as DANGGUI-SHAOYAO SAN, BUYANGHUANWU TANG,
TONGMAIYIZHI DAN and the like.
However, these Chinese medicines needs to be administered by a
herbalist doctor based on differential treatment that varies from
person to person, and cannot be used by the patients alone as they
are incapable of taking these Chinese medicines properly. The
effective ingredient ginsenoside Rg3 extracted from natural plants
of the present invention have high purity (80% or above 80%) and is
unique in curative effect, a prepared preparation is convenient to
use, patients can adjust the dosages according to self reactions by
themselves, and the effect of the ginsenoside after transdermal
absorption is moderate and lasting.
Panax ginseng C. A. Mey. is an araliaceae perennial herb and is
regarded as the top grade and described as that the Radix Ginseng
mainly reinforces internal organs, soothes mind and body
tranquilization, calms spirits, relieves palpitation due to fear,
improves eyesight, is uplifting, is beneficial to intelligence and
has the effects of reducing body weight and prolonging life after
being taken for a long time in the Chinese pharmaceutical ancient
book in regard to traditional Chinese medicine <Shen Nong's
Herbal Classic>. Modern medical research and chemical laboratory
analysis show that a chemical substance named as ginsenoside is
contained in the Radix Ginseng and has the obvious effects of
regulating the central nervous system of human, strengthening
heart, resisting fatigue, regulating substance metabolism and the
like, and therefore a good therapeutic effect is played on the
multiple diseases of the nervous system, the cardiovascular system,
the endocrine system and reproductive system.
The Radix Ginseng can regulate the balance of the excitation
process and inhibition process of the central nervous system. The
results of research of the influence on brain electrical activities
of animals of the Radix Ginseng show that: the Radix Ginseng
influences the two nerve processes of excitation and inhibition,
but mainly strengthen the excitation process of the cerebral
cortex. Due to the fact that the ginseng acts on the inhibition
process at the same time, the inhibition tends to concentration,
and differentiation is accelerated and is more complete. The Radix
Ginseng can regulate nerve functions and enable the disordered
nerve process caused by tension to be restored.
Some reports suggest that Radix Ginseng extract has an antagonism
effect on poor memory caused by anisodine and pentobarbital sodium,
can also improve memory consolidation impairment caused by
cycloheximide and sodium nitrite and memory reappearance deficits
caused by 40% ethyl alcohol. Radix Ginseng stem-leaf saponins are
injected into the abdominal cavity of rats in dosages of 200 mg/kg,
100 mg/kg and 50 mg/kg, the anisodine effect can be obviously
resisted, the memory of the rats can be obviously improved,
intracerebral RNA can be obviously increased, but no obvious effect
is produced on the DNA and protein content.
The Radix Ginseng also has obvious impact on cerebral blood flow
and cerebral energy metabolism. A Radix Ginseng preparation can
improve glucose intake of the rabbit brain, at the same time,
reduces the ratio of lactic acid, pyruvic acid and lactic
acid/pyruvic acid, and makes the utilization of glucose to transfer
to aerobic metabolism from an anaerobic metabolism pathway. The
Radix Ginseng also can make free inorganic phosphorus in the
cerebral cortex increase by 25%. The Radix Ginseng fruit saponin
can improve oxygen uptake ability of the brain. The total Radix
Ginseng saponin and total Radix Ginseng root saponin both have
protection against cerebral ischemia/reperfusion injury. In sum,
the Radix Ginseng can make animal brains more reasonably utilize
energy substance glucose, generates energy with oxidation, and
synthesizes more ATP for activity use such as learning, memorizing
and the like.
The ginsenoside Rg3 is a Radix Ginseng rare saponin, which is only
contained in radix ginseng rubra over more than 5 years for 3 parts
per 10 million, and is the most essential component in the Radix
Ginseng. The ginsenoside Rg3 has improving and preventing effects
on various diseases, and has a great therapeutic effect on elderly
common diseases, such as cardiovascular and cerebrovascular
diseases, coronary heart disease, limbs acratia, disability, memory
impairment.
The ginsenoside Rg3 is Radix Ginseng glycol tetracyclic
triterpenoid saponin, it is found in practical applications that
since the ginsenoside Rg3 has a larger molecular weight (785.02),
its lipid solubility and water solubility are both relatively poor,
making its oral absorption percentage being about 10.28%, and
resulting in relatively poor absorption of oral preparations,
results of animal pharmacokinetic experiments indicate that about
80% of the medicine in native form is not absorbed and is excreted
through feces, and the medicine is not fully used in the body,
which will influence full use of its clinical therapeutic effect.
Therefore, the ginsenoside Rg3 is made into topical preparations,
which directly enters the lymphatic system by absorption via neck
skin and posterior auricular mucosa, it is absorbed via the
blood-brain barrier so as to achieve a therapeutic effect on the
cerebral nervous system, and a better clinical therapeutic effect
is obtained and further confirmed through pharmacological
experiments and clinical applications.
Borneolum Syntheticum: studies of immunohistochemical staining
technique adopted by Zhao Baosheng, et al. show that Borneolum
Syntheticum can not induce expression of ICAM-1 on rat brain
microvascular EC, but can significantly reduce adhesion of
leukocytes and EC during brain trauma, and has a protective effect
on EC, the studies prove that the promotion of openness of the
blood-brain barrier with borneolum syntheticum is irrelevant to
ICAM-1, and borneolum syntheticum has a certain protective effect
on the rat brain tissue with brain trauma. Rats are divided into 12
groups by Ge Chaoli, et al., drenched with a borneolum syntheticum
paraffin oil solution 10, 13 mLkg, and are also divided into 1, 2,
4, 8, 24, 48 hours for each group according to the difference of
sampling time points, the control group is drenched with equal
amount of paraffin oil solution. The two groups of rats are
sacrificed and brains are taken at above-mentioned time points,
respectively, and observed with a transmission electron microscope.
The studies show that borneolum syntheticum has a significant
influence on tight junction between rat cerebral capillary
endothelial cells; compared with the control group, intercellular
tight junction gaps are widened, are discontinous, and are reduced
in structure, after drench with borneolum syntheticum for 4 hours,
changes start to occur, reach to peak at the 8.sup.th hour, and
return to normal at the 48.sup.th hour, which indicates that
borneolum syntheticum can make ultra microstructures of the rat
blood-brain barrier have reversible changes. The rabbit serum
containing traditional Chinese medicine borneolum syntheticum
prepared by Chen Yanming uses verapamil as positive control
medicine, it is observed and found with an MTT method that on MDCK
(Madin-Darby Canine Kidney) and HeLa (human uterine cancer) cell
lines, borneolum syntheticum can both significantly increase cell
toxicity caused by vincristine, and has a similar effect with
verapamil, which illustrates that borneolum syntheticum has a
significant inhibition effect on expression of P-glycoprotein.
There are also studies which show that promotion of physiological
openness of the blood-brain barrier with borneolum syntheticum is
due to the fact that after borneolum syntheticum enters the
blood-brain barrier, affinity of borneolum syntheticum with
P-glycoprotein is large, as a result, the borneolum syntheticum is
pumped out of cells, thus the vascular active substance 5-HT which
has small affinity with P-glycoprotein is accumulated in cells so
as to promote openness of the blood-brain barrier.
Styrax: rats are divided into a sulpiride combined storax group and
a single-use sulpiride group, after the rats continuously take oral
medication for one week, the surgery for installing probes in the
brain and neck of rats are performed, the equilibration is waited
for one hour, dialysate in the blood of right atrium of rats and
dialysate in intracerebral hippocampal tissues are collected at
each time period (30, 60, 90, 120, 150, 180 minutes) by blood
microdialysis and brain microdialysis methods, respectively, the
content of sulpiride in the sample is detected by a reversed-phase
high-performance liquid fluorescence chromatogram system, and the
content of sulpiride in the brain and the blood are compared after
medicine administration by a statistic method. As a result, it is
obvious that the concentrations of sulpiride in the brain and blood
of rats in the sulpiride combined storax group are higher than that
in the single-use sulpiride group, the concentration ratio of
sulpiride in the blood and brain of rats is 1:0.2 in the single-use
sulpiride group, the concentration ratio of sulpiride in the blood
and brain of rats is raised to 1:0.3 in the sulpiride combined
storax group, and compared with the single-use sulpiride group, the
concentration of sulpiride in the brain is raised by 39% and the
concentration of sulpiride in the blood is raised by 69% in the
sulpiride combined storax group. This indicates that the storax can
significantly increase the concentrations of sulpiride in the brain
and blood of rats, and the storax can facilitate the sulpiride to
penetrate the gastrointestinal barrier and the blood-brain
barrier.
Moschus: expert experiments indicate that: a Moschus aqueous
solution or suspension which is used for intravenous injection by
50 mg/kg or for lateral ventricle injection by 2.5 mg/kg can
de-synchronize the quiet and awake rabbit cortex
electroencephalogram (EEG) for a short time, and part of animals
are with restlessness behavior in a watch state, indicating that
the cerebral cortex can be excited and the cortical electrical
activity can be enhanced; the Moschus aqueous solution has an
obvious wake-up effect on anesthetized rabbits, lateral ventricle
injection is more effective than intravenous injection, and the
above specification may describe that the Moschus likely penetrates
the blood-brain barrier to directly act on the central nervous
system.
Rhizoma Acori Tatarinowii: rhizoma acori tatarinowii is one of
traditional resuscitation-inducing aromatic herbs, is singly used
among the people or is compatible with other traditional Chinese
medicines for wide usage, is mainly used for treating
encephalopathy and has a definite curative effect, and it prompted
that the rhizoma acori tatarinowii has a certain effect on the BBB.
Volatile oil is extracted by adopting the volatile oil extraction
method I described in the appendix I of the Chinese Pharmacopoeia
2005, the extraction rate is about 1.58%, and an oil removing water
decoction is concentrated to be 1 g/ml. A gas chromatography-mass
spectrometer (GC/MS) is adopted to detect that a volatile oil
sample contains .alpha.-asarone, .beta.-asarone, guaiene and
acorenone, the cerebrospinal fluid of a rat taking the volatile oil
is detected under the same conditions, and a result shows that the
.alpha.-asarone and the .beta.-asarone can penetrate the BBB to
enter the cerebrospinal fluid. A liquid chromatography-mass
spectrometer (HPLC/MS) is adopted to determine an oil removing
water decoction sample and the cerebrospinal fluid sample of a rat
drenched with the oil removing water decoction, three compounds are
detected, the molecular weights are 396, 452 and 339 respectively,
and a known rhizoma acori tatarinowii compound is not yet included
therein. The ultra microstructures of the cerebral cortex BBB of
the rats before and after medicine administration are observed
through the transmission electron microscope, it can be found that
all extracted parts of the rhizoma acori tatarinowii enable the
tight junction (TJ) of BBB endothelial cells to be loose, but the
BBB is basically complete and is not yet destroyed. Borneolum
syntheticum is used as a positive medicine to preliminarily discuss
the promoting effect on BBB passage of Evans blue (EB) of the
extracted parts of the rhizoma acori tatarinowii, namely the EB
content in the brain reflects the permeability of the BBB, the
statistical analysis shows that the EB content in the brain is
remarkably higher than that of a blank group (P<0.01) 2.5 hours
after the borneolum syntheticum is filled into the stomach, and the
self-administrating medicine with the rhizoma acori tatarinowii
dose of 11.7 g/kg does not produce an obvious effect within 0.5 to
2.5 hours; the administration time of the rhizoma acori tatarinowii
is prolonged, the EB content in the brain after 5 hours is
remarkably higher than that of the blank group (P<0.05) when the
volatile oil doses of the rhizoma acori tatarinowii are 23.4 g/kg
and 11.7 g/kg, and a dose dependency trend exists; when the total
dose is 11.7 g/kg, the EB content in the brain after 5 hours is
remarkably higher than that of the blank group (P<0.01). One
week after the extracted parts of the rhizoma acori tatarinowii are
filled into the stomachs of the rats, phenytoin sodium is injected
into the abdominal cavity, an HPLC method is adopted to detect the
concentration of the phenytoin sodium in the brain, and a result
shows that: the content of the phenytoin sodium in the brain of the
total-group animal taking the rhizoma acori tatarinowii is higher
than that in the brain of the animal group which does not take the
rhizoma acori tatarinowii, but a higher concentration (P<0.01)
is still kept after 4 hours. An HPLC-ECD is utilized to detect the
concentration of 5-hydroxytryptamine (5-HT) in brain tissues, and
statistical analysis shows that: compared with the blank group, the
content of the 5-HT in the brain of the rat in the volatile oil
group is increased (P<0.05) and the content of the 5-HT in the
rhizoma acori tatarinowii water decoction group and the content of
5-HT in the total medicine group are remarkably increased
(P<0.01). A HeLa cell line is applied to serve as a carrier for
evaluating the activity of the rhizoma acori tatarinowii to
P-glycoprotein on the BBB, and the parts (volatile oil, water
decoction and total medicine) of the rhizoma acori tatarinowii can
obviously strengthen the cytotoxicity of vincristine (VCR) after
0.5 hour (P<0.01); the volatile oil part can still obviously
strengthen the cytotoxicity of the VCR after 8 hours (P<0.01),
and the medicine effect maintaining time of the volatile oil is
obviously longer than that of the positive medicine verapamil (VER)
(P<0.01); the positive medicine and the parts of the rhizoma
acori tatarinowii have no obvious effect on the cytotoxicity of the
VCR after 24 hours (P>0.05). In sum, the rhizoma acori
tatarinowii can affect the ultra microstructure of the rat BBB,
strengthen the permeability of the BBB to the EB and promote
increase of the content of the CNS medicine phenytoin sodium
entering the brain and has the pharmacological actions of
activating the BBB and leading medicines to the brain. Action
mechanism research results show that the 5-HT content regulation by
the rhizoma acori tatarinowii is one of its BBB permeability
increase mechanisms, and in addition, the rhizoma acori tatarinowii
has a very strong effect of inhibiting exocytosis of the
P-glycoprotein medicine on the HeLa cell membranes, which is also
an action mechanism of BBB permeability increase.
Radix Curcumae: the curcumin contained in the Radix Curcumae has an
effect of inhibiting the senile dementia. The main cause of the
dementia is intertwining of amyloid .beta.. The curcumin enables
the intertwined amyloid .beta. to be loosened. The curcumin is a
powerful antioxidant and anti-inflammatory chemical molecule and
can penetrate the strictly guarded blood-brain barrier to
neutralize oxides and copper ions having oxidative toxicity, and
beyond that, the curcumin can further inhibit a cell transcription
factor (NF kappa B) causing inflammation. Therefore, the curcumin
is a key ingredient for protecting the brain tissues and decreasing
dementia. In India, the number of patients with dementia is only
ten percent of those in other countries, and curcumin's
contributions cannot go unnoticed.
CONTENTS OF THE INVENTION
The primary objective of the present invention is to provide the
performance and effect of 20(R)-ginsenoside Rg3 in preventing
or/and treating dementia and to provide new pharmaceutical uses of
20(R)-ginsenoside Rg3, namely new applications in medicines or
health foods for treating, coordinating and relieving dementia.
Another objective of the present invention is to provide a
ginsenoside Rg3 topical preparation for clinical use defects of
ginsenoside Rg3 such as poor oral absorption and poor blood-brain
barrier penetration. The ginsenoside Rg3 topical medicinal
preparation of the present invention is applied directly on both
sides of the nose, a neck and the skin behind ears, for treating
dementia, particularly treating and preventing Alzheimer's
disease.
To achieve the purpose, on one hand, the invention provides
applications of 20(R)-ginsenoside Rg3 in preparing a medicine or
health care product for preventing or/and treating dementia,
wherein the dementia is Alzheimer's disease and vascular dementia,
preferably Alzheimer's disease, and
wherein the medicine is composed of 20(R)-ginsenoside Rg3 and a
pharmaceutically acceptable carrier.
Particularly, the medicine further comprises one or more of Radix
Ginseng oil, Rhizoma Chuanxiong oil, Rhizoma Acori Tatarinowii oil,
Moschus oil or Radix Curcumae oil.
The 20(R)-ginsenoside Rg3 of the present invention can be used to
prevent or treat dementia either alone or in the form of a
medicinal composition containing the 20(R)-ginsenoside Rg3.
The medicine of the invention is administrated by oral, sublingual,
transdermal, intramuscular, subcutaneous, mucocutaneous and
intravenous ways.
The present invention provides a medicinal preparation for
preventing or treating dementia, with 20(R)-ginsenoside Rg3 used as
an active component, and corresponding pharmaceutical dosage forms.
The medicinal preparation uses the 20(R)-ginsenoside Rg3 as an
active component and comprises other pharmaceutically acceptable
carrier components,
wherein the content of 20(R)-ginsenoside Rg3 is equal to or greater
than 80%, preferably higher than 90%, and further preferably higher
than 95%.
The medicine of the present invention is present in forms of oral
preparation, injection and topical preparation. Wherein the oral
preparation comprises tablets, capsules, pills, powder, granules,
syrups or solutions; the injection comprises an injectable solution
or lyophilized powder for injection; and the topical preparation
comprises cream, ointments, sprays, aerosols, patches, gels,
naristillaes or cataplasms.
The medicine of the present invention is present in forms of
tablets, capsules, pills, powder, granules, syrups, solutions,
injections, sprays, aerosols, patches, gels, naristillaes or
cataplasms, namely medicinal preparations comprise tablets,
capsules, pills, powder, granules, syrups, solutions, injections,
sprays, aerosols, patches, gels, naristillaes, cataplasms and the
like, but are not limited to the forms above.
On the other hand, the present invention provides a medicine or
health care product containing 20(R)-ginsenoside Rg3 for preventing
or/and treating dementia.
Wherein the medicine or health care product further comprises one
of Radix Ginseng volatile oil, Moschus volatile oil, Rhizoma Acori
Tatarinowii volatile oil, Radix Curcumae volatile oil or Rhizoma
Chuanxiong volatile oil.
Particularly, the content of the 20(R)-ginsenoside Rg3 in the
medicine or health care product is .gtoreq.80%, preferably higher
than 90%, and further preferably higher than 95%.
The inventor employed advanced separation and purification
techniques to extract active component 20(R)-ginsenoside Rg3 for
preventing and treating dementia from medicinal Radix Ginseng
through considerable modern scientific research and carried out
anti-dementia pharmacodynamical and pharmacological studies on the
20(R)-ginsenoside Rg3 and its corresponding medicinal preparations,
results show that monomers of the 20(R)-ginsenoside Rg3 provide a
clear pharmacological action, high anti-dementia efficacy, a low
toxic or side effect and high safety, and a highly effective and
low toxic medicine is provided for anti-age and anti-dementia.
Compared with existing medicines for preventing and treating
dementia, the present invention has the following advantages:
1. The present invention explores new medicinal value of the known
compound 20(R)-ginsenoside Rg3, the 20(R)-ginsenoside Rg3 can be
used for preventing and treating dementia, and can be prepared into
medicines or health foods for preventing and treating dementia,
thus exploiting a new field for the application of medicinal Radix
Ginseng.
2. Series of experimental studies of the present invention
demonstrate that 20(R)-ginsenoside Rg3 has outstanding efficacy for
preventing and treating dementia. Ginsenoside Rg3 improves the AChE
positive nerve fiber density of the cerebral cortex significantly,
which shows that ginsenoside Rg3 has a protection effect on the
intracerebral cholinergic nerve system with Alzheimer's disease; in
experiments of the effect ginsenoside Rg3 on learning and memory of
a rat model with Alzheimer's disease, the time of swimming out of
rats in the water maze is short and the error time is less, which
implies that ginsenoside Rg3 can obviously improve the learning and
memory ability of the model rat with Alzheimer's disease;
ginsenoside Rg3 can reduce the .DELTA..beta. content in the
cerebrospinal fluid of patients with AD, and ginsenoside Rg3 can
effectively improve the patients' intelligence and memory and
improve the cognitive function and ability of daily living when
used in clinic treatment of AD.
3. 20(R)-ginsenoside Rg3 of the present invention has a strong
pharmacological effect, and has significant anti-aging efficacy,
quick action, a little toxic or side effect and good safety, and
20(R)-ginsenoside Rg3 can be taken chronically, and has good
medicinal prospect.
4. The product material of the present invention is abundant in
source, low in cost, safe in clinical use, and simple in
preparation process, and can be prepared into various preparations
and has small dosing and is easy to use, and thus easily
popularizing.
DESCRIPTION OF THE DRAWINGS
FIG. 1 shows secondary full-scan mass spectrums of quasi-molecular
ion [M-H]- of Ginsenoside Rg3 (A) and Dioscin (B);
FIG. 2 shows typical chromatograms of Ginsenoside Rg3 (channel I,
m/z 783.8.fwdarw.160.8) and internal standard Dioscin (channel II,
m/z 867.5.fwdarw.721.5) in liver homogenate determined by LC/MS/MS
methods; wherein (A) blank liver homogenates of rats; (B) adding 2
ng/ml Ginsenoside Rg3 and 100 ng/ml internal standard Dioscin to
the blank liver homogenates of rats; (C) cerebrospinal fluid sample
after administering orally 10 mg/kg Ginsenoside Rg3 to rats (10.8
ng/g).
DESCRIPTION OF THE EMBODIMENTS
The advantageous effects of the medicine of the present invention
are further illustrated by the following test examples; these
examples comprise pharmacodynamic tests of the medicine of the
present invention.
Test Example 1 Protection Effect of Ginsenoside Rg3 on
Intracerebral Cholinergic Nerve System of Rat Model with
Alzheimer's Disease
1. Material:
20(R)-ginsenoside Rg3 (purity>98%), produced by Dalian Fusheng
Natural Medicine Development Co., Ltd., with the batch number
being: 20120316; by comparison with a standard product provided by
National Institute for Food and Drug Control of China and by
measured by HPLC, the content accords with the calibration value,
and the measured value is 98.2%;
Ginsenoside Rg3 is prepared into a lotion for topical use according
to the formula and method of embodiment 7 for standby application;
the trial production of the lotion is conducted by Dalian Fusheng
Natural Medicine Development Co., Ltd., with the batch number
being: 20120425;
Positive medicine: Huperzine A, produced by Shanghai Hongqi
Pharmaceutical Factory, with the batch number being: 20111215;
D-galactose: produced by Shanghai Chemical Reagent No. 2 Factory,
with the batch number being: 20110607.
Ibotenic acid (IBO), produced by Sigma-Aldrich Co. LLC., with the
batch number being: 20101224.
60 preliminary elder female Wistar rats aged 15 months and having
body weights of 300-450 g, purchased from the Laboratory animal
center of Dalian Medical University, number of certificate of
quality: SCXK(13)2012-0003.
2. Method and Result:
2.1 Method
60 Wistar rats each of which having the body weight of 300-500 g
are randomly divided into 6 groups, with 10 in each group, and the
groups are named a preliminary elder normal control group, a
preliminary elder AD model group, a positive medicine Huperzine A
group, and high-dose, medium-dose and low-dose of Ginsenoside Rg3
topical preparation groups according to different treatment
methods, respectively. The rats are bred in separate cages, each
holding 4-5 rats, with natural lighting and random drinking and
feeding provided. Physiological saline is injected in the abdominal
cavity and grain of each rat in the preliminary elder normal
control group; Galactose intraperitoneal injection and IBO
intracerebral injection are given to all rats in the preliminary
elder AD model group, the positive medicine Huperzine A group, and
the high-dose, medium-dose and low-dose Ginsenoside Rg3 topical
preparation groups; while the rats in the Huperzine A group and the
high-dose, medium-dose and low-dose of Ginsenoside Rg3 topical
preparation groups are subjected to D-galactose injection for
modeling, rat of each group is given Huperzine A 0.3 mg/kg/d and
Ginsenoside Rg3 10, 5, 2.5 mg/kg/d respectively. Ginsenoside Rg3 is
applied to both sides of the nose of each rat of Ginsenoside Rg3
topical preparation groups. All the rats are subjected to the
medicines continuously for 6 weeks and then intracerebral bilateral
Meynert nuclei injection is carried out. Reference is made to a rat
cerebral stereotaxic atlas for Meynert nuclei localization, and
localization coordinates are AP-0.8 mm, Late 2.6 mm and DV 8.2 mm
During intracerebral injection, a rat is anesthetized with 3%
pentobarbital sodium (30 mg/kg) and then immobilized to a cerebral
stereotaxic apparatus, physiological saline dissolved IBO
(injection 1 .mu.l for each side containing 5 .mu.g of IBO) or 1
.mu.l normal saline is injected slowly into a Meynert nucleus, each
side takes injection for 5 minutes, and needle retaining for 10
minutes, wherein all the rats are subjected to cerebral perfusion
with 4% paraformaldehyde after modeling and are then immobilized
for 24 hours, and the brain is put in a sucrose phosphate buffer
until brain tissue blocks sink, the blocks are frozen in a
incubator and serially sectioned to 40 mm in thickness. The
sections are subjected to acetycholinesterase (AChE) histochemical
staining by methods recommended by Hedreen, et al. Observation is
given to the frontal lobe, the parietal lobe, the occipital lobe
and the entorhinal cortex (layer II) as well as parts in a
hippocampal CA1 region of the hippocampal formation such as a
stratum oriens, a stratum radiatum, a stratum lacunosum-moleculare
and a stratum moleculare of dentate gyrus for observing their AChE
positive nerve fiber densities, and the AChE positive nerve fiber
densities are counted with a number of intersections of positive
fibers with a standard grid by using a micrometer used for a
microscope.
2.2 Result
Results of acetycholinesterase positive nerve fiber density test
for the cortex and hippocampal formation in rats are seen in tables
1 and 2, all results are represented as (X.+-.S), and one-way
analysis of variance is carried out using an SAS 6.11 statistical
package. A significance level a is equal to 0.05.
TABLE-US-00001 TABLE 1 Rat cortical AchE positive nerve fiber
density (X .+-. S) Dosage N Frontal Parietal N Occipital N
Entorhinal Group (mg/kg/d) lobe/(10.sup.2 mm.sup.-2) lobe/(10.sup.2
mm.sup.-2) lobe/(10.sup.2 mm.sup.-2) cortex/(10.sup.2 mm.sup.-2)
Normal group 10 161.1 .+-. 16.5 179.4 .+-. 25.3 156.0 .+-. 15.7
164.4 .+-. 43.2 Model group 48 122.4 .+-. 20.9.sup.## 137.9 .+-.
30.0.sup.## 122.3 .+-. 6.7.sup.## 111.8 .+-. 12.9.sup.## Huperzine
group 0.3 122.1 .+-. 16.1.sup.## 108.7 .+-. 15.8.sup.##.DELTA.
131.6 .+-. 9.0.sup..DELTA. 132.4 .+-. 19.2.sup.# Ginsenoside Rg3 10
143.7 .+-. 22.1.sup..DELTA. 183.9 .+-. 24.9.sup.#**.degree. 167.2
.+-. 12.9.sup..DELTA..DELTA.**.degree. 161.1 .+-.
18.3.sup..DELTA..DELTA.**.degree. group 5 130.7 .+-.
20.8.sup..DELTA. 170.9 .+-. 21.2.sup.#** 160.2 .+-.
10.2.sup..DELTA..DELTA.** 142.6 .+-. 11.9 2.5 122.7 .+-.
18.6.sup..DELTA. 162.9 .+-. 22.0.sup.#** 145.2 .+-.
9.2.sup..DELTA..DELTA.** 130.2 .+-. 15.6 Compared with those of the
normal group, .degree.P > 0 05, .sup.#p < 0.05 and .sup.##p
< 0.01; compared with those of the model group, .sup..DELTA.p
< 0.05 and .sup..DELTA..DELTA.p < 0.01; compared with those
of the huperzine group, *p < 0.05 and **p < 0.01;
From the results in table 1, AChE positive nerve fiber densities
for all parts in the high-dose, medium-dose and low-dose of
ginsenoside Rg3 groups all gain a significant increase, and the
densities for both the frontal lobe and the occipital lobe are
significantly different (P<0.05 and P<0.01) from those in the
model group and the Huperzine group and are substantially restored
to a normal level (P>0.05, in comparison with that of the normal
group).
TABLE-US-00002 TABLE 2 Rat hippocampal formation AchE positive
nerve fiber density (X .+-. S) N Stratum N Stratum N Stratum N
Stratum lacunosum- moleculare of Dosage oriens/ radiatum/
moleculare/ dentate gyrus/ Group (mg/kg/d) (10.sup.2 mm.sup.-2)
(10.sup.2 mm.sup.-2) (10.sup.2 mm.sup.-2) (10.sup.2 mm.sup.-2)
Normal control 10 152.2 .+-. 18.9 134.2 .+-. 12.4 91.4 .+-. 10.8
141.1 .+-. 15.5 group Model control 48 125.3 .+-. 18.5.sup.# 103.4
.+-. 19.0.sup.## 73.1 .+-. 10.8.sup.# 112.4 .+-. 16.7.sup.## group
Huperzine 0.3 138.7 .+-. 20.1 121.1 .+-. 15.2 83.3 .+-. 9.6 120.0
.+-. 10.7.sup.# control group Ginsenoside 10 171.1 .+-.
16.9.sup..DELTA. 143.2 .+-. 20.5.sup..DELTA..DELTA. 99.2 .+-.
17.3.sup..DELTA. 142.7 .+-. 11.9 Rg3 group 5 153.0 .+-.
22.1.sup..DELTA. 135.1 .+-. 23.9.sup.#** 84.2 .+-.
10.9.sup..DELTA..DELTA.** 131.5 .+-. 14.7 2.5 140.1 .+-.
20.5.sup..DELTA. 122.4 .+-. 14.4.sup.#** 77.2 .+-.
9.1.sup..DELTA..DELTA.** 119.8 .+-. 12.4 Compared with those of the
normal group, .degree.P > 0.05, p < 0.05 and .sup.##p <
0.01; compared with those of the model group, .sup..DELTA.p <
0.05 and .sup..DELTA..DELTA.p < 0.01; compared with those of the
huperzine group, *p < 0.05 and **p < 0.01;
From the results in table 2, AChE positive nerve fiber densities
for all parts in the high-dose, medium-dose and low-dose
ginsenoside Rg3 groups are higher than those in the model group,
and the densities for the stratum oriens, stratum radiatum and
stratum moleculare of dentate gyrus in the CA1 region are not
significantly different (P>0.05 for all) from those in the
normal group.
The test results above show that ginsenoside Rg3 can protect the
intracerebral cholinergic nervous system of a rat model with
Alzheimer's disease.
Test Example 2 Effect of Ginsenoside Rg3 on Learning and Memory of
Rat Model with Alzheimer's Disease
1. Material:
20(R)-ginsenoside Rg3 (purity>98%), produced by Dalian Fusheng
Natural Medicine Development Co., Ltd., with the batch number
being: 20120316; by comparison with a standard product provided by
National Institute for the Control of Pharmaceutical and Biological
Products of China and measured by PHLC, the content accords with
the calibration value, and the measured value is 98.2%.
ginsenoside Rg3 is prepared into an oral emulsion (10 mg/ml)
according to the formula and method of embodiment 17 for spare; the
trial production of the oral emulsion is conducted by Dalian
Fusheng Pharmaceutical Co., Ltd., with the batch number being:
20120320;
Positive medicine. D-galactose (produced by Shanghai Chemical
Reagent No. 2 Factory), with the batch number being: 20110607.
Ibotenic acid (IBO), produced by Sigma-Aldrich Co. LLC., with the
batch number being: 20101224.
60 preliminary elder female Wistar rats aged 6 months and having
body weights of 180-250 g, purchased from the Laboratory animal
center of Dalian Medical University, number of certificate of
quality: SCXK(13)2012-0003.
2. Method:
Water maze pre-training is performed after feeding for 1 week under
the stable condition of laboratory, 60 rats with similar body
weights and good flexibility are selected, and are divided into 6
groups randomly, a normal control group, a sham operated control
group, a model control group, and high-dose, medium-dose and
low-dose ginsenoside Rg3 groups (10, 5, 2.5 mg/kg/d), wherein each
group has 10 animals.
The molding method applying intraperitoneal injection of 0.96%
D-galactose prepared by normal saline is applied to rats to make
model rat, 5 mL/kg, once daily for 6 continuous weeks. The rat of
sham operated control group are injected with equivalent normal
saline. The rat groups of the model control group and high-dose,
medium-dose and low-dose ginsenoside Rg3 groups are intracranially
injected with IBO starting from the 43.sup.th day, and each side is
slowly injected with 1 .mu.L of IBO, the sham operated group's rats
are injected with equivalent normal saline in the same encephalic
region (for 4 continuous weeks). The normal control group's rat are
without any processing and eat and drink normally. The rats of
high-dose, medium-dose and low-dose ginsenoside Rg3 groups are
administered intragastrically with ginsenoside Rg3 oral emulsion
daily while molding, and are administered for 4 w (weeks) after
molding.
The changes of animal behavioristics in each group are tested by
channel-type water maze test, and the learning and memory abilities
of rats are evaluated by number of entering blind ends and the time
needed to reach the terminal. All test results are expressed in
mean.+-.standard deviation, and the intergroup difference is
subjected to variance analysis by adopting SPSS10.0 statistical
package.
3. Result:
The test results are shown in table 3.
TABLE-US-00003 TABLE 3 Effect of Rg3 oral emulsion on learning and
memory of rat model with Alzheimer's disease (X .+-. S) Dosage
Swimming Error times Group (mg/kg/d) out time (s) (time) Normal
control group 10 18.26 .+-. 3.41 1.00 .+-. 0.75 Sham operated
control group 10 26.42 .+-. 5.04 1.65 .+-. 0.66 Model control group
10 59.88 .+-. 5.27* 5.44 .+-. 0.53* Ginsenoside Rg3 group 10 12.26
.+-. 3.26.sup.# 1.01 .+-. 0.41.sup.# 5 19.26 .+-. 3.23.sup.# 1.10
.+-. 0.44.sup.# 2.5 32.26 .+-. 3.21.sup.# 1.82 .+-. 0.43.sup.#
Compared with that of the normal group and the sham operated group,
*p < 0.01; compared with that of the model group, .sup.#p <
0.05
The shorter the time of animals swimming out of the water maze is
and the less the error times are, the greater the learning and
memory abilities of animals are. It can be seen from Table 3, the
time of swimming out and error times of animals of the model group
are obviously more than those of the normal group and sham operated
group, and the time of swimming out and error times of animals of
the high-dose, medium-dose and low-dose ginsenoside Rg3 groups are
obviously less than those of the model group, which indicates that
ginsenoside Rg3 can obviously enhance the learning and memory
abilities of model rats.
Test Example 3 Effect of Ginsenoside Rg3 on Cerebrospinal Fluid
.beta. Amyloid Protein Content of Patients with Alzheimer's
Disease
3.1 Test Material:
20(R)-ginsenoside Rg3 (purity>98%), produced by Dalian Fusheng
Natural Medicine Development Co., Ltd., with the batch number
being: 20120316; by comparison with a standard product provided by
National Institute for Food and Drug Control of China and measured
by PHLC, the content accords with the calibration value, and the
measured value is 98.2%;
ginsenoside Rg3 is prepared into a ginsenoside Rg3 oral solution
(10 mg/ml) according to the formula and method of Embodiment 27 for
standby application; the trial production of the ginsenoside Rg3
oral solution is conducted by Dalian Fusheng Natural Medicine
Development Co., Ltd., with the batch number being: 20120321;
positive control medicine, donepezil (5 mg*7, Eisai (China)
Pharmaceutical Co., Ltd.).
3.2 Test Method:
36 patients are all inpatients, and are randomly divided into a
ginsenoside Rg3 oral liquid group and a donepezil group, and each
group has 18 patients. The ginsenoside Rg3 oral liquid group is
orally administered ginsenoside Rg3 oral liquid (10 mg/d) before
sleeping; the donepezil group is orally administered 5 mg/d of
donepezil prior to sleeping. Both groups take medicine for 12
continuous weeks. 2 ml of cerebrospinal fluid is collected by
lumbar puncture, and is stored at -80.degree. C., the
.beta.-amyloid protein (A.beta.) content is determined by using
enzyme-linked immunosorbent assay (ELISA). A revised Hasegawa
dementia scale (HDS-R) is used to score, and the scores before and
after treatment are compared, increasing in scores means validness,
and unchanging or decreasing in scores means invalidness. The
enumeration data adopts .chi..sup.2 test, the comparison of
measurement data adopts t-test, all data is processed by adopting
SPSS12.0 statistical software. The test results are shown in table
4.
Analysis on the clinical therapeutic effect of the two groups. The
number of patients increased in scores of clinical symptom of the
ginsenoside Rg3 oral liquid group is 14, the number of patients
constant in scores is 3, and the number of patients decreased in
scores is 1, and thus the effective rate is 77.8%. The number of
patients increased in scores of clinical symptom of the donepezil
group is 13, and the number of patients constant in scores is 3,
the number of patients decreased in scores is 2, the effective rate
is 72.2%, and there is no significant difference between the two
groups (P>0.05).
TABLE-US-00004 TABLE 4 Comparison of scores of before and after
treatment and A.beta. Content of two groups (n = 18, X .+-. s)
Scores of clinical symptom A.beta. content Before Post Before Post
Group treatment treatment treatment treatment Ginsenoside 12.3 .+-.
5.2 18.7 .+-. 8.1.sup.#.DELTA. 5.9 .+-. 2.6 3.8 .+-. 1.5.sup.# Rg3
group Donepezil 13.5 .+-. 4.2.sup..DELTA. 17.3 .+-. 9.6.sup.# 5.3
.+-. 1.7 4.9 .+-. 1.2 control group Compared with the situation
prior to treatment within the same group: .sup.#P < 0.05 and
compared with the donepezil group: .sup..DELTA.P < 0.01.
It can be known from the results of table 4 that compared with the
situations before treatment, the scores of the two groups after
treatment are increased obviously (P<0.05), and the scores of
the ginsenoside Rg3 group are increased significantly (P<0.05).
The A.beta. content of the ginsenoside Rg3 group after treatment is
decreased obviously (P<0.05), while that of the positive
medicine control group after treatment shows no obvious changes
(P>0.05). Clinical observation shows that the application of
ginsenoside Rg3 in the clinical treatment of AD can effectively
enhance intelligence and memory of patents and improve their
cognitive functions and activities of daily living. The study
indicates that ginsenoside Rg3 can reduce the A.beta. content in
the cerebrospinal fluid of patents with AD. This may be one of its
action mechanisms of treating AD.
Test Example 4 Clinical Research on Effectiveness of Ginsenoside
Rg3 on Mild or Moderate Alzheimer's Disease
1. Test Material:
20(R)-ginsenoside Rg3 (purity>98%), produced by Dalian Fusheng
Natural Medicine Development Co., Ltd., with the batch number
being: 20120316; by comparison with a standard product provided by
National Institute for the Control of Pharmaceutical and Biological
Products of China and measured by PHLC, the content accords with
the calibration value, and the measured value is 98.2%;
ginsenoside Rg3 is prepared into a ginsenoside Rg3 naristillae (10
mg/ml) according to the formula and method of embodiment 29 for
standby application; the trial production of the ginsenoside Rg3
naristillae is conducted by Dalian Fusheng Natural Medicine
Development Co., Ltd., with the batch number being: 20120324.
2. Test Method:
89 patients with mild or moderate Alzheimer's disease are divided
into two groups randomly, one of which take ginsenoside Rg3, and
the other placebos. There are 46 patients in the group taking Rg3,
with 21 male and 25 female, aged from 58 to 87 (72.6.+-.6.8),
scoring from 10 to 24 (17.8.+-.2.3) points in the mini-mental state
examination (MMSE); there are 43 patients taking placebos, with 19
male and 24 female aged from 57 to 88 (71.8.+-.8.2), scoring from
10 to 24 (18.2.+-.2.7) points in MMSE. There are no obvious
differences between the ginsenoside Rg3 group and the placebo group
with respect to gender, age and MMSE scores. The patents in the
group taking ginsenoside Rg3 use the ginsenoside Rg3 naristillae
once a day for 12 continuous weeks, with 10 mg/ml for each
time.
For the patients taking placebos, except that what is used for
intranasal administration are placebos, the rest is conducted
according to the same administration method as that adopted by
those taking ginsenoside Rg3. The placebos are prepared according
to the method used to prepare the ginsenoside Rg3 naristillae in
embodiment 29. For them, except that ginsenoside Rg3 is not added,
the rest is identical to embodiment 29.
The ginsenoside Rg3 naristillae has a concentration of 10 mg/ml,
and colors, characters, tastes and dosages of the placebos are the
same as those of the ginsenoside Rg3 naristillae. The ginsenoside
Rg3 naristillae and the placebos are applied once a day for 12
continuous weeks. Examination is conducted once every four weeks
before and after medication, and the examination method is as
follows:
(1) MMSE Screening of AD: examining patients' cognitive functions
(orientation, memory, computational power, language competence,
ability in applying visual-spatial segment etc.; (2) Examining
patents' dementia degrees using the clinical dementia rating (CDR);
(3) Examining patents' activities of daily living using the
activity of daily living scale (ADL).
SPSS statistical software is adopted for examination results.
T-test is adopted before and after treatment, and it is also
adopted for the ginsenoside Rg3 naristillae group and the placebo
control group. For scoring results of MMSE, CDR and ADL of patients
with AD before treatment, see table 5
TABLE-US-00005 TABLE 5 Scoring results of MMSE, CDR and ADL of
patients with AD before treatment (x .+-. s, points) Group Number
MMSE CDR ADL Ginsenoside Rg3 group 46 17.8 .+-. 2.3 1.9 .+-. 0.3
47.2 .+-. 7.9 Placebo group 43 18.2 .+-. 2.7 2.0 .+-. 0.2 48.3 .+-.
6.1 Notes: when the group of ginsenoside Rg3 topical preparation is
compared with the placebo group, none of the differences among
MMSE, CDR and ADL has obvious significance (P > 0.05).
For scoring results of MMSE, CDR and ADL of patients with AD after
taking Rg3 and placebos for 12 weeks, see table 6.
TABLE-US-00006 TABLE 6 Scoring results of MMSE, CDR and ADL of
patients with AD after treatment for 12 weeks (x .+-. s, points)
Group Number MMSE CDR ADL Ginsenoside Rg3 46 24.1 .+-. 2.0** 1.2
.+-. 0.2* 40.5 .+-. 7.6** group Placebo group 43 18.7 .+-. 2.4 2.0
.+-. 0.2 49.5 .+-. 6.3 Notes: when the ginsenoside Rg3 naristillae
group is compared with the placebo group, *P < 0.05, and **P
< 0.01.
Cognitive functions (scored by MMSE): the curative effect of the
placebo control group indicates that after treatment, the
ginsenoside Rg3 group shows an obvious increase in its MMSE score
(P<0.01) when compared with the placebo control group; dementia
degrees (scored by CDR): after treatment, the ginsenoside Rg3
naristillae group shows a significant decrease in its CDR score
(P<0.05) when compared with the placebo control group;
activities of daily living (scored by ADL): after treatment, the
ginsenoside Rg3 naristillae group shows a very significant decrease
in its ADL score (P<0.01) when compared with the placebo control
group, and compared with the situation before treatment, the ADL
score after treatment is decreased very obviously by 7.1 points
(P<0.01).
In sum, the ginsenoside Rg3 is a safe and effective medicine used
for treating patients with AD, relieving mild and moderate
cognitive impairment, decreased self-care ability of daily living
and degree of dementia of patients with AD.
Test Example 5 Clinical Research on Effectiveness of Ginsenoside
Rg3 on Mild, Moderate Alzheimer's Disease
1. Test Material:
20(R)-ginsenoside Rg3 (purity>98%), produced by Dalian Fusheng
Natural Medicine Development Co., Ltd. with the batch number being:
20120316; by comparison with a standard product provided by
National Institute for the Control of Pharmaceutical and Biological
Products of China and measured by PHLC, the content accords with
the calibration value, and the measured value is 98.2%;
ginsenoside Rg3 is prepared into a ginsenoside Rg3 naristillae (10
mg/ml) according to the formula and method of embodiment 29 for
standby application; the trial production of the ginsenoside Rg3
naristillae is conducted by Dalian Fusheng Pharmaceutical Co., Ltd.
with the batch number being: 20120324.
2. Test Method:
99 patients with mild or moderate Alzheimer's disease, among which
47 are male, 52 are female, all aged from 55 to 85 (73.5.+-.7.6),
MMSE score is 1024 (16.3.+-.2.6) points.
Patients with AD all use the ginsenoside Rg3 naristillae once a
day, 10 mg/ml each time, and keep using it for 12 weeks.
Examination is conducted once every four weeks before and after
medication, and the medication method is as follows:
(1) MMSE screening of AD: examining patients' cognitive functions
(orientation, memory, computational power, language competence,
ability in applying visual-spatial etc.; (2) Examining patents'
dementia degree using the clinical dementia rating (CDR); (3)
examining patents' activities of daily living using the activity of
daily living scale (ADL).
SPSS statistical software is adopted for examination results.
T-test is adopted before and after treatment, and it is also
adopted for the ginsenoside Rg3 naristillae group and the placebo
control group.
For scoring results of MMSE, CDR and ADL of patients with AD, see
table 7.
TABLE-US-00007 TABLE 7 Scoring results of MMSE, CDR, ADL of
Patients with AD before and after treatment with Rg3 (x .+-. s,
points) Treatment for Scale name Before treatment Treatment for 4
weeks 12 weeks MMSE 16.3 .+-. 2.6 17.1 .+-. 1.9* 19.8 .+-. 1.7**
CDR 2.1 .+-. 0.3 1.9 .+-. 0.2 1.5 .+-. 0.2* ADL 49.6 .+-. 8.2 48.2
.+-. 7.9 42.5 .+-. 7.3** Notes: comparison is carried out on the
self-control group before and after treatment, *P < 0.05, and
**P < 0.01.
Cognitive functions (scored by MMSE): the effectiveness after
treatment for 12 weeks is significantly increased, the MMSE score
is increased by 3.5 points (P<0.01), and the MMSE score has been
increased obviously after the treatment for 4 weeks (P<0.05).
The degree of dementia (scored by CDR): the effectiveness after
treatment for 12 weeks is significantly decreased compared with
that of before the treatment, the CDR score is decreased by 0.6
points (P<0.05). The ability of daily living (scored by ADL):
the curative effect after treatment is obviously decreased compared
with that before the treatment, the ADL score is decreased by 7.1
points (P<0.01).
In sum, the ginsenoside Rg3 is a safe and effective medicine used
for treating patients with AD, relieving mild and moderate
cognitive impairment, decreased self-care ability of daily living
and degree of dementia of patients with AD.
The present invention will be described in further details with the
following embodiments.
Embodiment 1
1. Preparing raw materials according to the following formula
20(R)-ginsenoside Rg3 (98%) 2 g; azone 2 g; propylene glycol 10 ml,
and an appropriate amount of ethanol (65%);
2. Adding ginsenoside Rg3, azone, propylene glycol into ethanol
(65%), wherein the total volume after mixing is 85 ml;
3. Adding the mixture with a total volume of 85 ml into a 500 ml
flask, heating to 50-55.degree. C., stirring while maintaining the
temperature at 50-55.degree. C. until the 20(R)-ginsenoside Rg3 is
completely dissolved, stopping heating after stirring for 30
minutes, cooling to room temperature (15-25.degree. C.), and
supplementing ethanol (65%) to 100 ml to obtain a ginsenoside Rg3
liniment.
Embodiment 2
1. Preparing raw materials according to the following formula
20(R)-ginsenoside Rg3 (98%) 2 g; azone 2 g; polyethylene glycol 400
10 ml; and an appropriate amount of ethanol (70%);
2. Adding ginsenoside Rg3, azone, polyethylene glycol 400 into
ethanol (70%), wherein the total volume after mixing is 85 ml;
3. Adding the mixture (85 ml) into a 500 ml flask, heating to
50-55.degree. C., stirring while maintaining the temperature at
50-55.degree. C. until 20(R)-ginsenoside Rg3 is completely
dissolved, stopping heating after stirring for 30 minutes, cooling
to room temperature (15-25.degree. C.), and supplementing ethanol
(70%) to 100 ml to obtain a ginsenoside Rg3 lotion.
Embodiment 3
1. Preparing raw materials according to the following formula
20(R)-ginsenoside Rg3 (98%) 2 g; azone 2 g; polyethylene glycol 600
10 ml; and an appropriate amount of ethanol (70%);
2. Adding ginsenoside Rg3, azone, polyethylene glycol 600 into
ethanol (70%), wherein the total volume after mixing is 85 ml;
3. Adding the mixture (85 ml) into a 500 ml flask, heating to
50-55.degree. C., stirring while maintaining the temperature at
50-55.degree. C. until 20(R)-ginsenoside Rg3 is completely
dissolved, stopping heating after stirring for 30 minutes, cooling
to room temperature (15-25.degree. C.), and supplementing ethanol
(70%) to 100 ml to obtain a ginsenoside Rg3 lotion.
Embodiment 4
1. Preparing raw materials according to the following formula
20(R)-ginsenoside Rg3 (63%) 2 g; azone 2 g; polyethylene glycol 200
10 ml; and an appropriate amount of ethanol (65%);
2. Adding ginsenoside Rg3, azone, polyethylene glycol 200 into
ethanol (60%), wherein the total volume after mixing is 85 ml;
3. Adding the mixture with a total volume of 85 ml into a 500 ml
flask, heating to 50-55.degree. C., stirring while maintaining the
temperature at 50-55.degree. C. until 20(R)-ginsenoside Rg3 is
completely dissolved, stopping heating after stirring for 30
minutes, cooling to room temperature (15-25.degree. C.), and
supplementing ethanol (65%) to 100 ml to obtain a ginsenoside Rg3
lotion.
Embodiment 5
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (30%) 1 g, glycerol 2 g, polyethylene glycol
200 10 ml, and an appropriate amount of ethanol (70%);
2. Adding ginsenoside Rg3, azone, and polyethylene glycol 200 into
ethanol (70%), wherein the total volume after mixing is 85 ml;
3. Adding the mixture with a total volume of 85 ml into a 500 ml
flask, heating to 50-55.degree. C., stirring while maintaining the
temperature at 50-55.degree. C. until 20(R)-ginsenoside Rg3 is
completely dissolved, stopping heating after stirring for 30
minutes, cooling to room temperature (15-25.degree. C.), and
supplementing ethanol (70%) to 100 ml to obtain a ginsenoside Rg3
lotion.
Embodiment 6
1. Preparing raw materials according to the following formula
20(R)-ginsenoside Rg3 (98%) 1 g; Medium-Chain Triglyceride 20 g;
propylene glycol 10 ml; phosphatide 20 g; and an appropriate amount
of water for injection;
2. Adding ginsenoside Rg3, Medium-Chain Triglyceride, propylene
glycol, and phosphatide into a small amount of water for injection,
heating to 65-70.degree. C., stirring to dissolve ginsenoside Rg3,
adding the evenly stirred mixture into a homogenizer to be
homogenized, cooling to room temperature (15-25.degree. C.), and
supplementing water for injection to 100 ml to obtain a ginsenoside
Rg3 emulsion for injection.
The medium-chain triglyceride used in the embodiments of the
invention, i.e. decanoyl/octanoyl-glycerides, is a semisynthetic
natural functional lipid, which is safe and reliable according to
years of application at home and abroad and has broad application
field and use value, being abbreviated to MCT in Europe and
America. The product can be widely applied to medicine, food,
health care products, cosmetics and the like.
Embodiment 7
1. Preparing raw materials according to the following formula
20(R)-ginsenoside Rg3 (98%) 2 g; azone 2 g; Radix Ginseng oil 0.5
g; propylene glycol 10 ml; and an appropriate amount of ethanol
(65%);
2. Adding ginsenoside Rg3, azone, Radix Ginseng oil, and propylene
glycol into ethanol (65%), wherein the total volume after mixing is
85 ml;
3. Adding the mixture with a total volume of 85 ml into a 500 ml
flask, heating to 50-55.degree. C., stirring while maintaining the
temperature at 50-55.degree. C. until 20(R)-ginsenoside Rg3 is
completely dissolved, stopping heating after stirring for 30
minutes, cooling to room temperature (15-25.degree. C.), and
supplementing ethanol (65%) to 100 ml to obtain a ginsenoside Rg3
lotion.
Embodiment 8
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (98%) 2 g; azone 2 g; Radix Curcumae oil 1 g;
polyethylene glycol 400 10 ml; and an appropriate amount of ethanol
(70%).
2. Adding ginsenoside Rg3, azone, Radix Curcumae oil, and
polyethylene glycol 400 into ethanol (70%), wherein the total
volume after mixing is 85 ml;
3. Adding the mixture with a total volume of 85 ml into a 500 ml
flask, heating to 50-55.degree. C., stirring while maintaining the
temperature at 50-55.degree. C. until 20(R)-ginsenoside Rg3 is
completely dissolved, stopping heating after stirring for 30
minutes, cooling to room temperature (15-25.degree. C.), and
supplementing ethanol (70%) to 100 ml to obtain a ginsenoside Rg3
lotion.
Embodiment 9
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (98%) 2 g, azone 2 g, Moschus oil 0.05 g,
polyethylene glycol 600 10 ml, and an appropriate amount of ethanol
(70%);
2. Adding ginsenoside Rg3, azone, Moschus oil, and polyethylene
glycol 600 into ethanol (70%), wherein the total volume after
mixing is 85 ml;
3. Adding the mixture with a total volume of 85 ml into a 500 ml
flask, heating to 50-55.degree. C., stirring while maintaining the
temperature at 50-55.degree. C. until 20(R)-ginsenoside Rg3 is
completely dissolved, stopping heating after stirring for 30
minutes, cooling to room temperature (15-25.degree. C.), and
supplementing ethanol (70%) to 100 ml to obtain a ginsenoside Rg3
lotion.
Embodiment 10
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (63%) 2 g, azone 2 g, Rhizoma Chuanxiong oil
0.2 g, polyethylene glycol 200 10 ml, and an appropriate amount of
ethanol (65%);
2. Adding ginsenoside Rg3, azone, Rhizoma Chuanxiong oil, and
polyethylene glycol 200 into ethanol (65%), wherein the total
volume after mixing is 85 ml;
3. Adding the mixture with a total volume of 85 ml into a 500 ml
flask, heating to 50-55.degree. C., stirring while maintaining the
temperature at 50-55.degree. C. until 20(R)-ginsenoside Rg3 is
completely dissolved, stopping heating after stirring for 30
minutes, cooling to room temperature (15-25.degree. C.), and
supplementing ethanol (65%) to 100 ml to obtain a ginsenoside Rg3
lotion.
Embodiment 11
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (30%) 2 g, glycerol 2 g, Rhizoma Acori
Tatarinowii oil 0.2 g, polyethylene glycol 200 10 ml, and an
appropriate amount of ethanol (70%).
2. Adding ginsenoside Rg3, azone, Rhizoma Acori Tatarinowii oil,
and polyethylene glycol 200 into ethanol (70%), wherein the total
volume after mixing is 85 ml;
3. Adding the mixture with a total volume of 85 ml into a 500 ml
flask, heating to 50-55.degree. C., stirring while maintaining the
temperature at 50-55.degree. C. until 20(R)-ginsenoside Rg3 is
completely dissolved, stopping heating after stirring for 30
minutes, cooling to room temperature (15-25.degree. C.), and
supplementing ethanol (70%) to 100 ml to obtain a ginsenoside Rg3
lotion.
Embodiment 12
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (98%) 1 g, medium-chain triglyceride 20 g,
borneolum syntheticum 0.5 g, propylene glycol 10 ml, phosphatide 20
g, and an appropriate amount of water for injection; 2. Adding
ginsenoside Rg3, medium-chain triglyceride, borneol, propylene
glycol, and phosphatide to a small amount of water for injection,
heating to 65-70.degree. C., and stirring to dissolve the
ginsenoside Rg3, adding the evenly stirred mixture to a homogenizer
to be homogenized, cooling to room temperature (15-25.degree. C.),
and supplementing the water for injection to 100 ml to obtain a
ginsenoside Rg3 emulsion for injection.
Embodiment 13
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (98%) 2 g, azone 2 g, calculus bovis
artifactus 0.5 g, propylene glycol 10 ml, and an appropriate amount
of ethanol (65%);
2. Adding ginsenoside Rg3, azone, calculus bovis artifactitus, and
propylene glycol into ethanol (65%), wherein the total volume after
mixing is 85 ml;
3. Adding the mixture with a total volume of 85 ml into a 500 ml
flask, heating to 50-55.degree. C., stirring while maintaining the
temperature at 50-55.degree. C. until 20(R)-ginsenoside Rg3 is
completely dissolved, stopping heating after stirring for 30
minutes, cooling to room temperature (15-25.degree. C.), and
supplementing ethanol (65%) to 100 ml to obtain a ginsenoside Rg3
lotion.
Embodiment 14
1. Preparing raw materials according to the following ratio
20(R)-Ginsenoside Rg3 (98%) 2 g, azone 2 g, styrax 0.5 g,
polyethylene glycol 400 10 ml, and n appropriate amount of ethanol
(70%);
2. Adding ginsenoside Rg3, azone, styrax, polyethylene glycol into
ethanol (70%), wherein the total volume after mixing is 85 ml;
3. Adding the mixture with a total volume of 85 ml into a 500 ml
flask, heating, stirring for 30 minutes to dissolve the mixture,
cooling naturally, and supplementing 70% ethanol to 100 ml to
obtain the a ginsenoside Rg3 topical preparation.
Embodiment 15
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (98%) 1 g, soybean oil 20 g, propylene glycol
10 ml, phosphatide powder 20 g, and an appropriate amount of
distilled water;
2. Adding ginsenoside Rg3, soybean oil, propylene glycol, and
phosphatide powder to a small amount of distilled water, heating to
65-70.degree. C., and stirring to dissolve the ginsenoside Rg3,
adding the evenly stirred mixture to a homogenizer to be
homogenized, cooling to room temperature (15-25.degree. C.), and
supplementing the distilled water to 100 ml to obtain a ginsenoside
Rg3 oral emulsion.
Embodiment 16
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (98%) 1 g, soybean oil (for injection) 50 g,
medium-chain triglyceride 100 g;
2. Heating ginsenoside Rg3 (98%), soybean oil, medium-chain
triglyceride to 80-85.degree. C., and stirring to dissolve
ginsenoside Rg3 to obtain a ginsenoside Rg3 injection.
Embodiment 17
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (98%) 1 g, Radix Ginseng volatile oil 2 g,
propylene glycol 10 ml, Rhizoma Chuanxiong oil 0.5 g, phosphatide
20 g, and an appropriate amount of distilled water;
2. Adding ginsenoside Rg3, Radix Ginseng volatile oil, propylene
glycol, Rhizoma Chuanxiong oil and phosphatide to a small amount of
distilled water, heating to 65-70.degree. C., and stirring to
dissolve the ginsenoside Rg3, adding the evenly stirred mixture to
the homogenizer to be homogenized, cooling to room temperature
(15-25.degree. C.), and supplementing the distilled water to 100 ml
to obtain a ginsenoside Rg3 oral emulsion.
Embodiment 18
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (purity 85%) 1 g, Rhizoma Acori Tatarinowii
oil 20 g, propylene glycol 10 ml, phosphatide powder 20 g, and an
appropriate amount of distilled water;
2. Adding ginsenoside Rg3, Rhizoma Acori Tatarinowii oil, propylene
glycol, and phosphatide powder to a small amount of distilled
water, heating to 65-70.degree. C., and stirring to dissolve the
ginsenoside Rg3, adding the evenly stirred mixture to a colloid
mill to be treated, cooling to room temperature (15-25.degree. C.),
and supplementing the distilled water to 100 ml to obtain a
ginsenoside Rg3 oral emulsion.
Embodiment 19
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (purity 63%) 1 g, tulip oil 10 g, and
medium-chain triglyceride 100 g;
2. Heating ginsenoside Rg3, tulip oil, and medium-chain
triglyceride to 50-55.degree. C., and stirring to dissolve
ginsenoside Rg3 to obtain a ginsenoside Rg3 oral liquid.
Embodiment 20
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (purity 30%) 1 g, Rhizoma Chuanxiong oil 10
g, medium-chain triglyceride 100 g;
2. Heating ginsenoside Rg3, Rhizoma Chuanxiong oil, medium-chain
triglyceride to 50-55.degree. C., and stirring to dissolve
ginsenoside Rg3 to obtain a ginsenoside Rg3 oral liquid.
Embodiment 21
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (purity 98%) 1 g, Moschus oil 2 g, propylene
glycol 10 ml, phosphatide 20 g, and an appropriate amount of
distilled water.
2. Adding ginsenoside Rg3, Moschus oil, propylene glycol, and
phosphatide to a small amount of distilled water, heating to
65-70.degree. C., stirring to dissolve the ginsenoside Rg3, adding
the evenly stirred mixture to a homogenizer to be homogenized,
cooling to room temperature (15-25.degree. C.), and supplementing
the distilled water to 100 ml to obtain a ginsenoside Rg3 oral
emulsion.
Embodiment 22
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (purity 98%) 1 g, medium-chain triglyceride
20 g, Moschus oil 3 g, propylene glycol 10 ml, phosphatide 20 g, an
appropriate amount of water for injection.
2. Adding ginsenoside Rg3, medium-chain triglyceride, Moschus oil,
propylene glycol, and phosphatide to a small amount of water for
injection, heating to 65-70.degree. C., stirring to dissolve the
ginsenoside Rg3, adding the evenly stirred mixture to a homogenizer
to be homogenized, cooling to room temperature (15-25.degree. C.),
and supplementing the water for injection to 100 ml to obtain a
ginsenoside Rg3 emulsion for injection.
Embodiment 23
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (purity 98%) 1 g, medium-chain triglyceride
20 g, Rhizoma Acori Tatarinowii oil 2 g, polyethylene glycol 100 10
ml, phosphatide 20 g, and an appropriate amount of water for
injection.
2. Adding ginsenoside Rg3, medium-chain triglyceride, Rhizoma Acori
Tatarinowii oil, propylene glycol, and phosphatide to a small
amount of water for injection, heating to 65-70.degree. C.,
stirring to dissolve the ginsenoside Rg3, adding the evenly stirred
mixture to a homogenizer to be homogenized, cooling to room
temperature (15-25.degree. C.), and supplementing the water for
injection to 100 ml to obtain a ginsenoside Rg3 emulsion for
injection.
Embodiment 24
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (purity 98%) 1 g, sucrose 50 g, Rhizoma Acori
Tatarinowii oil 2 g, dextrin 100 g, and silicon dioxide 5 g;
2. Taking the formulated amount of Rhizoma Acori Tatarinowii oil,
and silicon dioxide, and evenly mixing; making silicon dioxide
fully adsorb Rhizoma Acori Tatarinowii oil; adding ginsenoside Rg3,
sucrose, and dextrin; granulating with 70% ethanol, sieving, and
drying at 65-70.degree. C. to obtain ginsenoside Rg3 granules.
Embodiment 25
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (purity 98%) 1 g, sucrose 50 g, Rhizoma
Chuanxiong oil 2 g, dextrin 100 g, and silicon dioxide 3 g;
2. Taking the formulated amount of Rhizoma Chuanxiong oil, and
silicon dioxide, and mixing evenly, making silicon dioxide fully
adsorb Rhizoma Chuanxiong oil; adding ginsenoside Rg3, sucrose, and
dextrin; granulating with 70% ethanol, sieving, and drying at
65-70.degree. C. to obtain ginsenoside Rg3 granules
Embodiment 26
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (purity 98%) 1 g, starch 100 g, Radix Ginseng
oil 3 g, microcrystalline cellulose 100 g, .beta.-cyclodextrin 10
g, and talcum powder 1 g;
2. Taking the formulated amount of .beta.-cyclodextrin, adding 100
ml of water to dissolve the .beta.-cyclodextrin, maintaining the
temperature at 70.degree. C. with a water bath, adding Radix
Ginseng oil, stirring the mixture for 30 minutes; taking out the
solution, cooling, and filtering the precipitate to obtain an
inclusion compound of Radix Ginseng oil. adding ginsenoside Rg3,
starch, and microcrystalline cellulose, granulating with an
appropriate amount of ethanol; sieving, milling, adding talcum
powder, and performing the mixture to obtain ginsenoside Rg3
tablets
Embodiment 27
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (purity 98%) 1 g, sucrose 50 g, Rhizoma
Chuanxiong oil 3 g, .beta.-cyclodextrin 10 g, sodium benzoate 2 g,
and an appropriate amount of distilled water;
2. Taking the formulated amount of .beta.-cyclodextrin, adding
water to dissolve .beta.-cyclodextrin, maintaining the temperature
at 70.degree. C. with a water bath, adding Rhizoma Chuanxiong oil,
stirring the mixture for 30 minutes; taking out the solution,
cooling, and filtering the precipitate to obtain an inclusion
compound of Rhizoma Chuanxiong oil. Adding ginsenoside Rg3,
sucrose, and sodium benzoate, and adding the distilled water to 500
ml to obtain ginsenoside Rg3 oral liquid.
Embodiment 28
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (purity 98%) 1 g, Moschus oil 1 g, 90%
ethanol 10 ml, glycerol 5 g, an appropriate amount of F12/F114
(propellant of (Freon) compound). The total amount of all raw
materials is 100 g;
2. Weighing accurately the formulated amount of ginsenoside Rg3,
and Moschus oil, adding into ethanol to dissolve ginsenoside Rg3
and Moschus oil, then adding glycerol, mixing evenly to prepare a
solution, subpackaging the solution with aerosol containers,
mounting valves, rolling tight sealing caps, and then filing mixed
propellant F12/F114.
Embodiment 29
1. Preparing raw materials according to the following ratio
20(R)-ginsenoside Rg3 (purity 98%) 1 g, Rhizoma Chuanxiong oil 1 g,
ethanol (60%) 10 ml, polyethylene glycol 200 5 ml, glycerol 20 g,
and an appropriate amount of water for injection;
2. Weighing accurately ginsenoside Rg3 (1 g), and Rhizoma
Chuanxiong oil (1 g), adding 10 ml of 60% ethanol, 5 ml of
polyethylene glycol 200, and 20 g of glycerol, heating the mixture
at a low temperature, stirring to dissolve the mixture, and adding
the water for injection to obtain a ginsenoside Rg3
naristillae.
Orifice-opening medicinal used in the embodiments of the invention,
such as Radix Ginseng (volatile) oil, Rhizoma Chuanxiong (volatile)
oil, Moschus (volatile) oil, Radix Curcumae (volatile) oil, Rhizoma
Acori Tatarinowii (volatile) oil, borneolum syntheticum, calculus
bovis artifactus, tulip oil and styrax, are mainly effective in
opening the orifices with herbal aromatics and inducing
resuscitation and are clinically used to treat diseases such as
stroke and delirium. Studies of recent years discover that inducing
resuscitation drugs used to treat brain diseases by affecting the
blood-brain barrier (BBB), some of the resuscitation-inducing drugs
can promote permeability of other medicines so that these drugs
exert their main acting mechanism of inducing resuscitation to
restore consciousness, and it is speculated that the mechanism may
be the theoretical basis of heart meridian distribution of these
medicines. For example, borneolum syntheticum can improve
blood-brain barrier permeability and promote medicines to enter
brain tissues, and the resuscitation-inducing drugs such as
Moschus, benzoin and styrax have some opening action on the
blood-brain barrier for rats under physiological conditions and can
promote medicines to enter brain tissues.
During preparation of Rg3 preparations, components such as azone,
polyethylene glycol, ethanol and traditional Chinese medicine
volatile oils are added into each prescription, the addition of
these components enables the blood-brain barrier to open so that
Rg3 can enter brain tissues and exert anti-dementia action, and the
present invention also uses "Detection test for ginsenoside Rg3
medicines of different dosage forms absorbed through blood-brain
barrier upon administration in embodiment 30" to verify again that
Rg3 has entered the brains of the rats under physiological
conditions. Therefore, all of the prescriptions and processes
designed in the invention and prepared samples have an
anti-dementia action.
The Radix Ginseng oil used in the embodiments of the invention is
purchased from Jiangxi Ji'an City Qingyuan District Hualong Spice
Oil Co., Ltd.; the Rhizoma Chuanxiong oil is purchased from Jiangxi
Ji'an City Senhai Spice Co., Ltd.; the Moschus oil is purchased
from Jiangxi Baicao Pharmaceutical Co., Ltd.; the Rhizoma Acori
Tatarinowii is purchased from Xi'an Ruiying Biotechnology Co.,
Ltd.; the tulipa oil is purchased from Jiangxi Bencao Natural Spice
Oil Co., Ltd.; the Radix Curcumae oil is purchased from Jiangxi
Hengcheng Natural Spice Oil Co., Ltd.; the Radix Ginseng volatile
oil is purchased from Jiangxi Huanqiu Natural Spice Co., Ltd.
The Radix Ginseng oil comprises main components: an essential oil
.gtoreq.11% in content and <30 KOHmg/g in acid value, a
ginsenoside, ginseng polysaccharides, a volatile oil, amino acids
and polypeptides, as well as .beta.-elemene, .beta.-gurjunene,
.beta.-panacene, .alpha.-panacene, caryophyllene, .beta.-farnesene
and palmitic acid; the Radix Ginseng volatile oil comprises main
components: sesquiterpenes about 40%, and oxygen compounds and
long-chain alkanes.
Embodiment 30 Detection Test for Ginsenoside Rg3 Medicines of
Different Dosage Forms Absorbed Through Blood-Brain Barrier Upon
Administration
1. Test Material
1.1 Animal
SD rats, 50 days old, male, about 260 g in body weight, clean
animal, purchased from the Laboratory animal center of Dalian
Medical University, laboratory animal license NO: SCXK (Liao)
2004-0017.
1.2 Instrument and Reagent
Rat cerebrospinal fluid suction apparatus: large and small surgical
scissors, tweezers, hemostats, and syringes (1 mL); a graduated
scale, trays, aseptic cotton balls, iodophor, Angle Two brain
stereotaxic apparatus (by Paxinos Atlas USA Myneurolab company), a
low speed dental drill (by Grobet File Co. Of America Inc), a
micro-injection pump (by American company of KD Scientific Inc),
and 10 .mu.L, and 100 .mu.L, microsyringes (by American company of
Hamilton Inc).
Instrument for measuring Rg3 medicine content in rat cerebrospinal
fluid: API type-4000 triple quadrupole tandem mass spectrometer,
fitted with an ion-spray ion source and an Analysis 1.3 data
processing system, by American company of Applied Biosystems Inc;
Agilent 1100 high-performance liquid chromatography infusion pump,
an autosampler, by Agilent Technologies Co. Ltd. Chromatographic
columns Nucleosil C18 columns (50*4.6 mm I.D., 5 .mu.m in particle
size), by Dalian Elite Analytical Instruments Co., Ltd.
1.3 Instrument and Medicine
Ginsenoside Rg3 preparations prepared in embodiments 1-2,
embodiments 6-14, embodiments 16-21 and embodiments 25-29.
2. Test Method
(1) Administration of Different Dosage Forms of Ginsenoside Rg3
1) Oral administration: an oral preparation of ginsenoside Rg3 is
given to SD rats by lavage, the medicines of all high-dosage,
medium-dosage and low-dosage groups are: 20, 10 and 5 mg/kg (the
dosage above is pure dosage of Rg3)/bid, respectively, and each
dosage group has 18 rats;
2) External administration: ginsenoside Rg3 is given to SD rats, an
external medicine is applied to dampness-eliminating Touqiao
acupoint or the both sides of nose, neck and back of ear, the
medicines of all high-dosage, medium-dosage and low-dosage groups
are: 10, 5, and 2.5 mg/kg (namely amounts of Rg3 are respectively
10, 5 and 2.5 mg/kg)/bid, respectively, and each dosage group has
18 rats;
3) Administration by injection: an intravenous injection of
ginsenoside Rg3 at the tail of rat is given to SD rat, wherein the
high-dosage, medium-dosage and low-dosage groups respectively are:
6, 3, 1.5 mg/kg/bid, and each dosage group has 18 rats.
(2) Rat Cerebrospinal Fluid Suction
Rat cerebrospinal fluid is sucked after orally, topically and
injection administering different preparations of ginsenoside Rg3
for 4 hours. The rat cerebrospinal fluid is sucked by using a
method of scarifying endorhachis under direct vision, referring to
a method of a document (Improvement on a suction method of the rat
cerebrospinal fluid. Journal of Hebei Medical University, 2010
(31)2: 125-127). Each rat is anesthetized with 10% chloral hydrate
by intraperitoneal injection, the head of the rat is fixed by brain
straight body positioning ear rods and front tooth rods, the
occipital region is sheared and sterilized, and a longitudinal
incision (about 2 cm) is cut along the posterior midline, and the
muscles at the dorsal part of napex are bluntly separated with a
pair of scissors. The bottommost layer muscles attached to the
occipital bone L are scraped away by the back of a scalpel to avoid
bleeding, the wound is washed with distilled water after exposing
atlanto-occipital fascia, after cleaning is carried out with a
sterile cotton ball, the endorhachis is longitudinally scarified
with a scalpel by 0.3 cm, and the flow of the clear cerebrospinal
fluid can be seen, a 100 .mu.l microsyringe (round head) prepared
in advance is placed at the crevasse to suck the cerebrospinal
fluid, each rat is sucked for 2 times, and 100 .mu.l for each
suction; the cerebrospinal fluids of 3 rats in each dosage group
are mixed evenly to be prepared into one sample, and 6 biological
samples are prepared in each dosage group.
The rats without being administered with ginsenoside Rg3 are blank
control.
(3) Determination of Ginsenoside Rg3 Content in Cerebrospinal Fluid
of Rats Administered with Different Dosage Forms
The medicine concentrations in the rat cerebrospinal fluid are
determined by LC/MS/MS methods.
Medicine:
ginsenoside Rg3 standard reference substance, purchased from
National Institute for the Control of Pharmaceutical and Biological
Products of China, with the batch number being: 110804-200603.
ginsenoside Rg3 raw material, produced by Dalian Fusheng Natural
Medicine Development Co., Ltd., with the batch number being:
2012303, and the content is 98.2% after HPLC calibration of
ginsenoside Rg3 standard reference substance;
the content determination method of the high-dosage, medium-dosage
and low-dosage ginsenoside Rg3 groups in different preparations of
other ginsenoside Rg3 is the same as the calibration method of
ginsenoside Rg3 raw material, and the test results of the content
of ginsenoside Rg3 of different preparations are shown in the test
results in detail.
Reagent:
acetonitrile, methanol, ethyl acetate are all chromatographic pure
reagents, and are purchased from Thermo Fisher Scientific Inc. and
Tianjin Kangke Deke Science and Technology Ltd. respectively; the
saline solution is purchased from Shenyang Zhiying pharmaceutical
plant.
Tissue Sample Processing
Taking 200 .mu.l of cerebrospinal fluid after sucking, adding 100
.mu.l of methanol solution, 100 .mu.l of internal standard solution
(100 ng/ml dioscin and methanol solution) and 0.5 ml of water
respectively and mixing uniformly, adding 3 ml of ethyl acetate,
eddying for 1 minute, oscillating for 10 minutes, and centrifuging
(4000 rpm) for 8 minutes, separating the supernatant and
blow-drying under air flow of 40.degree. C., dissolving the residue
in 200 .mu.l moving phase and taking 20 .mu.l for analysis.
Chromatographic condition: the moving phase is
methanol-acetonitrile-10 mmol/l ammonium acetate solution
(47.5:47.5:5, v/v), the flow rate is 0.7 ml/min, and the column
temperature is room temperature.
Mass spectrum condition: ionic propulsion voltage: -3500 V;
pressure of gas 1 within the source (GS1, N2): 30 p.s.i.; pressure
of gas 2 (GS2, N2): 30 p.s.i.; pressure of curtain gas (N2): 15
p.s.i.; pressure of collision gas (N2): 3 p.s.i.; temperature
within the source: 500.degree. C. Detection mode: negative ion
detection; Scanning manner: the reaction monitoring (MRM) manner is
selected, and the ionic reactions for qualitative analysis are m/z
783.8.fwdarw.m/z 160.8 (ginsenoside Rg3), m/z 867.5.fwdarw.m/z
721.5 (internal standard, dioscin) respectively. The corresponding
secondary full-scan mass spectrum is shown in FIG. 1.
Specificity of the Method
The cerebrospinal fluid of blank rat without being administrated
with medicine is taken, besides no internal standard solution
addition and then additional addition of 100 .mu.l methanol, the
balance is operated under "tissue sample treatment" item to obtain
the chromatogram of the blank sample; the ginsenoside Rg3 standard
reference substance solution (2 ng/ml) and internal standard
solution (100 ng/ml) are added to the blank rat cerebrospinal fluid
to obtain corresponding chromatogram according to the same
operation; the chromatogram of the cerebrospinal fluid sample of
rat after administrating is obtained according to the same
treatment.
FIG. 2 shows the chromatograms of the blank sample of the rat
cerebrospinal fluid, blank sample having adding Rg3 standard
reference substance and internal standard (dioscin) solution and
sample after administrating.
The results show that endogenous substances in the tissues do not
interfere the determination of ginsenoside Rg3 and the internal
standard dioscin.
Preparation of Working Curve
Taking homogenate supernatant 200 .mu.l of rat tissue of the blank
control group, and adding the ginsenoside Rg3 reference series
solution 100 .mu.l (adding the 100 .mu.l of Rg3 reference solution
to the 2000 of supernatant) to prepare tissue samples with the
equivalent tissue concentrations of 2, 4, 10, 20, 40, 100, 200 and
400 ng/g. Except for the addition of 100 .mu.l of methanol,
operating under the standard of "tissue sample processing" to
establish the working curve. Regression calculation is performed
with the weighted least squares method by taking the analyte
concentration as the abscissa value, and taking the peak area ratio
of analyte to the internal standard spectrum as the ordinate value,
to obtain a linear regression equation which is the working
curve.
According to the working curve, the linear range of the measured
concentration of ginsenoside Rg3 is: 2-400 ng/g, with the minimum
of 2 ng/g. Working curve: y=0.0014+0.0091x (r=0.9992).
Simultaneously quality control samples (QC samples) of high, medium
and low concentrations are made, and two-sample analysis performed
on each concentration. The concentration of QC samples is
calculated according to the working curve of each analysis batch,
and at most 2 out of 6 QC (total of 18 samples) samples with
different concentrations are allowed to exceed 15% of the
theoretical value (the lowest point at 20%), otherwise this batch
data is not accepted.
Sample Measurement of Different Preparations
Operation is carried out under the standard of "tissue sample
processing", to prepare a working curve for each analysis batch,
and the measured results of the cerebrospinal fluid samples of
administered rats are shown in table 8.
Data Processing
Medicine concentration data in the cerebrospinal fluid of each
administered rat is given out respectively, and the average value
and standard deviation are calculated.
TABLE-US-00008 TABLE 8 Content of ginsenoside Rg3 in cerebrospinal
fluid of rats during BBB test for different preparations High dose
Content of Rg3 in concentrations the cerebrospinal Preparations
Characteristics of dosage forms (mg/kg) fluid (x .+-. s, ng/g)
Embodiment 1 Topical application, different 10 6.8 .+-. 2.1
transdermal and solubilization excipients Embodiment 2 Topical
application, different 10 8.1 .+-. 3.2 transdermal and
solubilization excipients Embodiment 6 Emulsion for injection,
phosphatide 6 19.5 .+-. 4.3 excipients Embodiment 7 Topical
application, Radix Ginseng 10 30.2 .+-. 11.1 oil BBB promoters
Embodiment 8 Topical application, Radix 10 32.1 .+-. 13.0 Curcumae
oil BBB promoters Embodiment 9 Topical application, Moschus oil 10
35.0 .+-. 13.9 BBB promoters Embodiment Topical application,
rhizoma 10 30.9 .+-. 13.5 10 chuanqiong oil BBB promoters
Embodiment Topical application, Rhizoma Acori 10 30.2 .+-. 14.0 11
Tatarinowii oil BBB promoters Embodiment topical application,
borneolum 10 20.8 .+-. 7.8 12 syntheticum BBB promoters Embodiment
Topical application, calculus bovis 10 22.2 .+-. 7.5 13 artifactus
BBB promoters Embodiment Topical application, styrax BBB 10 23.0
.+-. 6.9 14 promoters Embodiment Emulsion for injection, soybean
oil 6 20.0 .+-. 5.3 16 excipients Embodiment Oral emulsion, Radix
Ginseng 20 26.2 .+-. 8.2 17 volatile oil BBB promoters Embodiment
Oral emulsion, Rhizoma Acori 20 21.5 .+-. 6.0 18 Tatarinowii BBB
promoters Embodiment Oral emulsion, tulip oil BBB 20 23.3 .+-. 4.9
19 promoters Embodiment Oral emulsion, rhizoma chuanqiong 20 25.6
.+-. 5.4 20 oil BBB promoters Embodiment Oral emulsion, Moschus oil
BBB 20 22.0 .+-. 6.8 21 promoters Embodiment Granules, Rhizoma
Chuanxiong oil 20 19.5 .+-. 7.9 25 BBB promoters Embodiment
Tablets, Radix Ginseng oil BBB 20 18.0 .+-. 6.1 26 promoters
Embodiment Oral solution, rhizoma chuanqiong 20 21.8 .+-. 5.2 27
oil BBB promoters Embodiment Aerosol, Moschus oil BBB 10 36.9 .+-.
14.6 28 promoters Embodiment Naristillae, rhizoma chuanqiong oil 10
45.1 .+-. 16.4 29 BBB promoters
The measurement results in table 8 show that through the medicine
concentration of ginsenoside Rg3 measured in the cerebrospinal
fluid of rats administered with the different dosage forms
(typical) of the ginsenoside Rg3 developed by the present
invention, the various dosage forms of the present invention can
all penetrate the blood-brain barrier into the encephala to play a
therapeutic effect.
* * * * *